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Abstract: The silk films were prepared by dissolving degummed Bombyx mori raw silk fibers in LiBr salt solution using double distilled water as a solvent and subjecting the solution to dialysis using cellulose tube. These films may be of some use in treating burn injuries. We have characterized these films using X-rays and other physical methods to obtain information about the structure-property relation. We have also compared the parameters of silk films with that of silk fibers.

Key words: Silk Fibers, silk films, X-ray Diffraction, burn injuries.

INTRODUCTION

The silk fibroin is used to prepare fibers, films, nano particles, thin strips and patches. Our earlier research works is on the silk fibers,[1,2] Silk is a natural protein produced by the domestic as well as wild silk worms. The amino acid composition of silk fiber of Bombyx mori mainly consists of glycine, alanine and serine. These three simple amino acids form the crystalline regions of silk fiber, while the amino acids with polar side chains form the amorphous regions. The silk fiber is partly crystalline and partly amorphous. Bombyx more fibers are composed of fibrous proteins with fibroin core (72–81%) and a surrounding glue protein Sericine (19–28%). The silk protein has a molecular mass of around 300 kDa and it is a linear polypeptide. [3, 4].

Silk fiber is used on daily basis as surgical suture for several centuries due to its good biological properties including bio-compatibility and low inflammatory reaction [5]. Further silk fibers have characteristic properties like good water vapour and oxygen permeability [6, 7], blood compatibility [8], accelerated collagen formation and proliferation of cultured human skin fibroblasts [9, 10]. It also has an easy control of structural conformation [11]. Silk has been applied in a wide variety of biomedical applications such as drug delivery systems [12] and enzyme immobilization [13]. For drug delivery, especially silk protein drugs, silk materials exhibits high encapsulation efficiency and controllable drug release kinetics due to control of crystalline beta-sheet formation [14].

In this work we have prepared pure silk film from raw fibers using simple solution casting method and its characterization has been carried out using X-ray diffraction and FTIR analysis. We have also carried out the anti-microbial activity test for the obtained silk fibroin in its solution form.

EXPERIMENTAL PART

Materials and Method:

Bombyx mori silkworm fresh silk reel were collected from local Central Silk Board at Mysore. All other chemicals used here were purchased from Merk India.

Preparation of Silk solution:

Silk solution was prepared according to the protocol described in similar studies. Briefly, fresh Silk fibers (8g/100ml) of Bombyx mori were degummed by boiling in 0.2 M Na2CO3 solution for 30 min. The degummed silk fibers were washed with water to remove residual Sericine and then air dried. The dried silk is immersed in 9.3M LiBr solution at 80oC to produce silk solution. This solution was then dialyzed against deionized water for 48 h using dialysis cellulose tube (MWCO 12 kDa, Sigma) to remove LiBr salt. The silk solutions (approximately 5%) were obtained after filtration and then stored at 4oC. This Solution was cast on glass plates at room temperature for 5-6days. After drying, carefully we have removed the film from glass plates, and were stored in a desicator for further use [15]. Figure 1 shows the photographs of silk fiber and the silk films including the dialysis setup. (Figure 1)

X-ray Diffraction Studies:

The X-ray diffraction patterns for the silk films and silk fibers were recorded using Righaku Denki Miniflex II Desktop Diffractometer, with Cu-Kα radiation for the 2θ range of 60 -700 with scanning speed of 50/min and step size of 0.020. These measurements were done at room temperature and care was taken to avoid mechanical distortions. Figure 2 show recorded X-ray patterns.

FT- IR analysis:

Secondary structure and conformation of the silk films and silk fibers were measured by using Fourier Transform Infrared (FTIR) Spectroscopy (Perking -Elmer Spectra GX USA) keeping air as reference. In this analysis resolution 4 cm-1 was chosen and data collection was in the range 1900 - 700 cm-1. Figure 3 shows the recorded FTIR spectrum for film and fibers

Antimicrobial activity by Well-Diffusion Method

The Silk fibroin solution was tested for antimicrobial activity by well-diffusion method against E.coli and Bacillus subtilis. The pure cultures of organisms were sub cultured in nutrient broth at 35°C on a rotary shaker at 200 rpm. Wells of 6-mm diameter were made on nutrient agar plates using gel puncture. Each strain was swabbed uniformly onto the individual plates using sterile cotton swabs. Using a micropipette10 μL, 20 μL and 30 μL of the sample of silk fibroin solution was added onto each of wells on all plates and positive control used Chloramphenicol [16, 17]. Figure 5 Shows.

RESULTS AND DISCUSSIONS

The X-Ray Diffraction profile obtained for the Silk fibers (a), degummed silk fibers (b) and the silk film is shown in Figure 2. The profile obtained shows that there is no such changes in the diffraction peaks obtained for silk fibers and silk film, but it is seen that the silk film is more amorphous as compared to the silk fibers. We have determined the average crystallite size and the lattice strain in these samples by employing W-H plot method [18] and also the percentage crystallinity was calculated. The obtained results from these calculations do support that the silk fiber is more crystalline than that of the silk film. The calculated results are tabulated in Table 1. Pair correlation study was also carried out using the XRD patterns. Figure 3 shows the pair correlation function for three different samples. Values of pair correlation function decreases with the formation of film and the disorder sets in the region of distances 5 to 6 Angstroms. (Table 1, Figure 2 and 3 should be placed as possible after this paragraph)

The FTIR spectra obtained for silk fibers and silk film show the same absorption spectrum and functional groups confirming the purity of the silk film prepared here. The spectral scan between 1900 cm-1 and 700 cm-1 is considered for the prediction of amide groups in silk protein. The silk is usually characterized by the β sheet absorption peaks which is found around 1630, 1530 and 1240 cm-1 and an α-helix absorption peaks around 1655 cm-1[15]. From the obtained spectra shown in Figure 4, it is seen that the absorption peaks lies in the range of 1625-1630cm1(amide I),1520-1530 cm-1 (amide II) and 1265-1270 cm-1 (amide III),which are the characteristic absorption peaks of β-sheet which confirms the Bombyx Mori silk fibers and silk film. (Figure 4 should be placed below this)

From the test for its antimicrobial activity, after incubation at 35°C for 24 hours, there was no zone of inhibition was observed. This result indicates that the obtained silk fibroin solution did not show any antimicrobial activity against tested organisms. Since silk is a chain of proteins, it does not possess any property of resisting the growth of microbes on its own. But further it can be made antimicrobial by adding antimicrobial agents to it. Thus for treating burn injuries these silk films should be added with the antimicrobial agents and the required drugs before the formation of films.

CONCLUSIONS

We have prepared silk films in the laboratory using solution casting method. It is evident from the studies carried out here that crystallinity and crystallite size decreases with the formation of silk films. Values of pair correlation function decreases with the formation of film and the disorder sets in the region of distances 5 to 6 Angstroms. In FTIR studies, the observed absorption peaks do support the beta-pleated arrangements of the chains. The Antimicrobial activity test shows that the silk fibroin solution prepared does not show any opposition to the growth of microbes, but further it can be used for treating in desired applications by adding and drawing these silk fibroin films with the drug to fulfill our requirements. Further work is in progress.

Acknowledgements

Authors thank UGC India, for Funds to the University of Mysore through UPE & CPEPEA Projects.

Table 1. The Lattice parameters obtained for the silk fibers and the Silk film